Allergic responses to commonly encountered substances in the everyday environment, or hypersensitivity reactions, are commonplace, with millions of Americans suffering from some form of hypersensitivity. Such allergic reactions are also often associated with the existence of asthma, a common disorder of the airways that is estimated to affect 4 to 5 percent of the population of the United States. There are no known cures for allergies or asthma, and currently available treatments in most cases only alleviate symptoms.
The human lung mast cell is a critical effector of respiratory hypersensitivity responses characteristic of both allergies and asthma. The release of inflammatory mediators from lung mast cells plays a central role in the pathophysiology of human allergic disorders. However, attempts to study the biochemistry and pharmacology of human lung mast cells has been stymied by technical difficulties in purification, low yields, inconsistent responsiveness to IgE-mediated stimulation, and short survival in vitro of these cells.
Methods to purify human lung mast cells are extremely difficult to execute and only a few laboratories worldwide have been successful at providing purified preparations of these cells.
Schulman et al. (J. Immunol. 1982 129:2662-2667) describe a method for purification of human lung mast cells where human lung tissue is finely minced and then dispersed into a single cell suspension using a series of enzymes. However, the cells purified by this method remain viable in culture for only short-term (24-96 hours).
Attempts have been made to co-culture human lung mast cells with fibroblasts and human tumor cell lines to extend the time period during which they are viable. Human lung mast cells cultured in culture dishes coated with human fibroblasts survived for approximately 15-30 days during which time the mast cells did not purify and did not proliferate (Levi-Schaffer et al. J. Immunol. 1987 139(2):494-500; and Dvorak et al. Am. J. Pathol. 1991 139(6):1309-18) while human lung mast cells cultured in dishes coated with a human tumor line survived for approximately 30 days (Hartzell et al. ARRD 139(4):A119, 1989). The precise mechanisms and chemical effectors through which the feeder cells allowed prolonged survival of mast cells was not determined in any of these studies.
Also, it is not known whether human lung mast cells in these co-cultures remain fully differentiated and maintain their unique characteristics manifested in vivo. Specifically, it is not known whether the critical cellular processes associated with mediator synthesis, storage and release remain unaltered under these culture conditions and thus maintain their specific phenotype as human lung mast cells. It is known that these mast cells do not proliferate under these conditions.
Attempts have also been made to raise mast cells from CD34 positive precursor stem cells. Most commonly, the source of these cells is human fetal cord blood, but peripheral blood and fetal liver have also been used. The ability to grow mast cells followed the cloning, in the early 1990's of stem-cell factor. Key to the success was combining stem cell factor with specific cytokine growth factors; usually interleukin-6 and/or Interleukin-3 surprisingly, addition to this mast cell culture system of interleukin-4, the cytokine that most defines allergic immunity, induces programmed cell death (apoptosis)(Oskeritzian et al. J. Immunol. 1999 162:5105-5115). Thus, the combination of growth factors mandatory for cultivating precursor-derived mast cells is critical. Although mast cells obtained by this method appeared to be useful for studies of areas such as mast cell biochemistry and signal transduction, their comparability to human lung mast cells was not conclusively demonstrated. Some reports have questioned the full maturity of such cells, with up to 50% of the nuclei in these cells displaying atypia which may be indicative of aberrant development (Bischoff et al. Proc. Natl. Acad. Sci. USA 1999 96:8080-8085; Dvorak et al. J. Leukoc. Biol. 1993 54:465-485; Toru et al. J. Allergy & Clin. Immunol. 1998 102:491-502). Furthermore, these cells and mature human lung mast cells responded differently to several physiologic and pharmacologic agonists. For example, human cord blood-derived mast cells can produce IL-13 only under co-stimulation with anti-IgE and stem cell factor, whereas human lung mast cells require only anti-IgE to produce IL-13. The cells also exhibit disparate responses to adenosine. Adenosine inhibits IgE-mediated histamine release in cord-blood precursor-derived mast cells as opposed to enhancement of anti-IgE-induced release observed in freshly isolated human lung mast cells(Kanbe et al. Int. Arch. Allergy Immunol. 1999 119(2):138-142; Suzuki et al. Biochem. Biophy. Res. Commun. 1998 242:697-702; Peachell et al. Am. Rev. Respir. Dis. 1988 138(5):1143-1151).
Bischoff et al. (Proc. Natl. Acad. Sci. USA 1999 96:8080-8085) described enzymatic dispersion methods for intestinal mast cell purification from human intestinal surgery specimens. However, fundamental differences in the biology of mast cells originating in different tissues and organs of the human body have been well documented. For example, basic differences in triggers, surface receptors and mediators released from skin mast cells versus lung mast cells are remarkable. These major differences render it impossible to directly apply methods for culturing viable mast cells derived from one organ such as the intestine to the culturing of mast cells from another organ such as the lung (Schulman, E. S. and D. G. Raible. 1998. In: Pulmonary Diseases and Disorders, vol. 1, 3rd edition, A. P. Fishman (ed.), McGraw Hill: New York, pp. 289-301; Schulman, E. S. Crit. Rev. Immunol. 1993 13:35-70).